Anti c kit

Immunostaining was performed as described previously^{9 (link)}. E10.5 embryos were fixed (2% paraformaldehyde/PBS, 20 min, 4 °C), equilibrated in 20% sucrose/PBS at 4 °C overnight and then embed in the Tissue Tek before freezing. From each WT and KO embryo, we sequenced the sections and chose 4-5 sections per embryo from ~100 sections at the similar area along the rostral-caudal axis. After additional blocking of endogenous biotin step, antibodies staining were performed. Primary and secondary antibody were used at the following concentrations: Anti-c-Kit (1:100, BD), biotinylated anti-CD34 (1:200, eBioscience), Runx1 (1:500, Abcam) and Hoechst for nuclear staining. Additionally, Ncl (1:100), GFP (MBL, 1:200), RFP (Rockland, 1:200), Lamp1 (Abcam, 1:100) or p62 (Abcam, 1:400) were used for staining ECs and pre-HSCs sorted by flow cytometry and spun onto slides. Secondary antibodies: Alexa Flour 647 anti-rat IgG (1:500), AF488 anti-rabbit IgG (1:1000) and Streptavidin Cy3 (1:1000). The image procedures were performed by confocal microscope (Zeiss LSM 880).

Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized in 0.1% Triton X-100/PBS, and then preincubated with blocking solution consisting of PBS containing 5% normal goat serum and 0.05% Tween-20. Cells were then incubated for 2 h in primary antibody [mouse anti-CD133, anti-C-kit, anti-vWF, anti-CDH5 (VE-cadherin), anti-α-SMA, or anti-PDGFRβ (BD Pharmingen, San Diego, CA)]. Reactions were visualized by FITC/TRITC-conjugated anti-human secondary antibody (Vector Laboratories, Burlingame, CA). All samples were observed with a fluorescence microscope (Olympus, Tokyo, Japan).

BM-MSC phenotypes were analyzed by flow cytometry using a FACSCalibur (BD Biosciences, NJ, USA). A total of 1 × 10⁶ cells/mL were incubated with FITC-, PerCP-, PE-, or APC-conjugated anti-CD34, anti-CD45, anti-CD29, anti-CD44, anti-CD90, anti-c-kit, or anti-Sca-1 antibodies (BD Biosciences) in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) for 30 minutes at room temperature.
Adipogenic differentiation was induced by culture with adipogenic medium supplemented with 10⁻⁸M dexamethasone and 10⁻⁴M L-ascorbic-acid-2-phosphate (Sigma-Aldrich, St. Louis, MO) as previously described (Ngoc et al., 2011) [32 (link)]. After 30 days of induction, cells were fixed in 3% formaldehyde in PBS for 10 minutes and stained with Oil Red O.
Osteogenic differentiation was induced by incubation with culture medium that consisted of ascorbic acid, dexamethasone, 6-glycerol phosphate (Sigma-Aldrich, St. Louis, MO), and calcium deposits which were visualized by alizarin red.

The aspirated blood was collected into a 1.5 mL tube per mouse. Then, the femur and tibia from the left limb of each mouse were dissected and transferred to a 15 mL conical tube containing Hanks’ balanced salt solution supplemented with 1% fetal bovine serum (FBS). PBMCs and BM-KSL cells, which are stem cell populations of EPCs, were isolated as previously reported [29 (link)].
Lineage-negative BM cells (BM-Lin-) were incubated with saturating concentrations of directly labeled anti-c-Kit (dilution, 1:25; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Sca-1 antibodies (at 1:25 dilution) (BD Biosciences) for 30 min on ice. Afterward, BM-KSL cells were isolated by live sterile cell sorting using a BD FACSAria™ Fusion flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).